How to detect the 2019 novel coronavirus?

(This article was originally published on January 23, 2020.) 

On January 21, 2020, the U.S. Centers for Disease Control and Prevention (CDC) reported the first case in the United States of the new coronavirus emerged in Wuhan, China last month. The arrival of new SARS-like coronavirus in U.S. heightens concerns about global spread after the infection cases reported in Thailand, Japan, South Korea and Taiwan. Meanwhile, a panel of Chinese health experts confirmed that the new virus is able to spread between people, which indicates it could be much harder to control.

Via next generation sequencing (NGS) of cultured virus or samples from several pneumonia patients, the etiologic agent responsible for the pneumonia cases in Wuhan has been identified as a novel betacoronavirus (in the same family as SARS-CoV and MERS-CoV). Electron microscopy revealed the virus is a coronavirus with a characteristic crown morphology.

The genome sequence of this betacoronavirus is crucial to develop specific diagnostic tests and to identify potential intervention options. Full genome sequence data from the viruses have been released and are available on NCBI (https://www.ncbi.nlm.nih.gov/nuccore/MN908947). Working directly from sequence information, a few laboratories developed several genetic amplification (PCR) assays to detect the novel coronaviruses. A few diagnostic kits developed by commercial companies, using the real-time PCR (RT-PCR) assay have been put to use rapidly. The timely emergence of detection methods is also one reason for the sharp rise in the number of confirmed cases.

Principle of novel coronavirus detection

The virus detection method can be basically illustrated as below.

Step 1: Isolate the novel coronavirus from the patients and sequence its genome.

 Step 2: Compare the genome sequence of coronavirus with human genome to find out the specific sequence in the virus genome.

Step 3: Design PCR amplification primers and fluorescent probe primers for detecting the specific sequences identified in step 2.

Step 4: Extract RNA from suspected individual’s serum, and convert the RNA into cDNA. The cDNA is then used as template and mixed with the PCR primers and probes for amplification. If the fluorescence signal increases rapidly and Ct value is less than 37, it can be determined as positive; If there’s no fluorescence detected, or the fluorescence signal grows slowly and Ct value finally ends up above 40, it can be determined as negative.

Resources

WHO guidance

On 17 January 2020, WHO provided interim guidance to laboratories and stakeholders involved in laboratory testing of patients who meet suspected case of pneumonia associated with the 2019 novel coronavirus.

• Laboratory testing for 2019 novel coronavirus (2019-nCoV) insuspected human cases.

Available protocols

Here are some protocols already have some degree of validation for laboratories wishing to test for coronavirus. It is strongly recommended to contact the protocol provider institution if you have any questions, or unusual lab findings.

• Diagnostic detection of Wuhan coronavirus 2019 by real-timeRT-PCR – Charité, Berlin Germany (pdf)

• Detection of 2019 novel coronavirus (2019-nCoV) in suspectedhuman cases by RT-PCR – Hong Kong University (pdf)

• China CDC Primers and probes for detection 2019-nCoV